The Degradome: Proteolytic enzymes, substrates, inhibitors, and regulatory proteins in health and disease

Proteolysis is an irreversible posttranslational modification that affects every single cell of an organism.
Over the last decades proteolytic enzymes have been identified to be master switches in the regulation of the immune system, in neuronal development and neurodegeneration,
and in apoptosis and cancer progression. Substrates define protease roles. Therefore, the identification of single enzymes, their substrates and inhibitors,
the “protease web”, is crucial to understand complex molecular events responsible for pathophysiological conditions, and subsequently also important for drug development.

1A) Oligomeric structure of human meprins

1B) Cleavage specificity of meprin a and meprin ß

Click on an image for a larger representation

Figure 1A: Oligomeric structure of human meprins

(A) Meprin α and meprin β are multi-domain enzymes, building dimers
linked by one intermolecular disulphide bond between the MAM (meprin A5 protein tyrosine phosphatase µ-like) domains. Meprins are expressed as zymogens with
a propeptide (PRO) N-terminal to the protease domain (CAT) that must be cleaved off proteolytically to gain full activity. Only meprin α contains an inserted
domain (I) which is cleaved by furin during the secretory pathway, resulting in secretion of the protein and further oligomerisation. Meprin α is the largest
secreted protease known, up to 6 mDa in size, as visualized by electron microscopy of purified recombinant enzyme. Meprin β predominantly remains membrane
bound but can be shed from the cell surface by ectodomain shedding through ADAM10 and ADAM17 activity. (B) Crystal structure of the ectodomain
of dimeric human meprin β in different orientations (pdb accession numbers 4GWN, 4GWM). One monomer is displayed with a transparent surface, revealing the ribbon
structure. (C) Model of the membrane bound form of meprin β and its orientation at the cell surface. For structural details see (Arolas et al., 2012, PNAS).

Figure 1B: Cleavage specifity of meprin α and meprin β

(A-D) With the help of proteomics approaches based on peptide libraries and
native substrates, the cleavage specificity of human meprins has been determined. Results are displayed in the WebLogo style, where the most preferred amino acid
residues are shown for positions P6 to P6’. Both proteases prefer negatively charged amino acids at the P1’ position. For detailed information see (Becker-Pauly
et al., 2012, Mol Cell Proteomics). (E) Incubation of fluorogenic substrates consisting of only aspartate and glutamate residues demonstrates
the ability of meprin β to cleave completely acidic peptides. (F) The unique specificity of meprin β is based on structural features of the active
site cleft. Positively charged arginine (Arg) residues (dark blue) can interact with negatively charged amino acid residues of the substrate.

Proteomics analyses enabled the identification of more than 100 new meprin substrates and
revealed a possible link to neurodegeneration regarding the release of Aβ peptides by meprin β. Cleavage of pro-inflammatory cytokines by meprins and genetic
studies demonstrated contribution of these proteases to the progression of inflammatory bowel disease. This was further validated in DSS induced colitis in
meprin α and meprin β knockout mice.

2) Proteolytic interactions responsible for the shedding of APP by meprin β(Click on image for a larger representation)

Figure 2: Proteolytic interactions responsible for the shedding of APP by meprin β

Importantly, we discovered meprin α and meprin β as procollagen proteinases, capable of cleaving off
the globular C- and N-terminal prodomains of fibrillar collagen type I and type III (Broder et al., 2013, PNAS; Kronenberg et al., 2010, J Invest Dermatol). This proteolytic
process is sufficient to induce collagen fibril assembly as visualized by transmission electron microscopy. The biological relevance was demonstrated with the help of meprin
α and meprin β knock-out mice, which exhibit decreased collagen deposition in skin resulting in impaired tensile strength. On the other hand, overexpression of meprin
metalloproteases was found under fibrotic conditions in skin (keloids) and lung (pulmonary hypertension). Thus, regulation of meprin activity by specific inhibition to reduce
collagen maturation might be a suitable approach for the treatment of certain pathological conditions.

Further studies, including appropriate animal models, will elucidate the precise molecular pathways mediated by meprin α and meprin β activity in health and disease. The
generation of specific meprin inhibitors is a crucial goal to develop potential therapeutics for the treatment of meprin-associated pathologies.

3) De novo fibrillogenesis of type I collagen
after cleavage by meprin α or meprin β / In vivo analysis of collagen I maturation, deposition and mechanical strength in skin of wt, meprin α, and meprin β knockout mice
(Click on image for a larger representation)

Figure 3: De novo fibrillogenesis of type I collagen after cleavage by meprin α or meprin β / In vivo
analysis of collagen I maturation, deposition and mechanical strength in skin of wt, meprin α, and meprin β knockout mice

De novo fibrillogenesis of type I collagen after cleavage by meprin α or meprin β.(A) Transmission electron micrographs of negatively stained collagen fibrils assembled after cleavage of recombinant procollagen type I heterotrimer by meprin α and meprin β.
100 µg/ml of recombinant procollagen I was incubated in reaction buffer with either 15 nM BMP-1 (+ PCPE-1 equimolar to the substrate), meprin α or meprin β in a total volume of 10 µl at 37°C
for 60 min. As a control, untreated recombinant procollagen I was visualized. (B) Cartoon summarizes procollagen processing by different proteases and subsequent assembly of
collagen fibrils.

In vivo analysis of collagen I maturation, deposition, and mechanical strength in skin of wt, meprin α, and meprin β knockout mice.(C) Azan-stained skin cross-sections picture collagen deposition (red arrow) in the dermis of Mep1a-/- and Mep1b-/- mice compared to the skin of age-matched
wild-type animals. Scale bar = 200 μm.
(D) Dermal collagen fibrils examined by transmission electron microscopy. Fibrils in Mep1a-/- and Mep1b-/- skin cross-sections display a less tightly packed organization
(red arrows) while the wt collagen fibrils show the characteristic compact and uniform arrangement. Scale bar = 1 μm. (E) Dermal collagen deposition was quantified by light microscopy.
The mean thickness of the different biopsies was obtained by averaging five measurements per section (***p<0.001). (F) Box plot shows the average diameters (nm) for dermal collagen fibrils
of Mep1a-/-, Mep1b-/-, and wt mice, obtained by measuring 414 fibrils from 3 wt mice, 598 fibrils form 3 Mep1a-/- mice, and 414 fibrils from 3 Mep1b-/- mice. Bottom of the box indicates
the 25th percentile, top the 75th percentile. The median values are shown as horizontal lines, indicating the 50th percentile. Whiskers indicate the lowest datum still within 1.5 interquartile
range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile. Outliers are shown as open circles. (***p<0.001). (G) Determination of the maximum
tensile strength of the skin of meprin α and meprin β knockout mice revealed a significant decrease (***p<0.001) compared to the skin of wild-type mice. Adapted from Broder et al., 2013, PNAS.